Quantitative methods to precisely measure cellular states in vivo have become increasingly important and desirable in modern biology. Recently, stimulated Raman scattering (SRS) microscopy has emerged as a powerful tool to visualize small biological molecules tagged with alkyne (C≡C) or carbon–deuterium (C–D) bonds in the cell-silent region. In this study, we developed a technique based on SRS microscopy of vibrational tags for quantitative imaging of lipid synthesis and lipolysis in live animals. The technique aims to overcome the major limitations of conventional fluorescent staining and lipid extraction methods that do not provide the capability of in vivo quantitative analysis. Specifically, we used three bioorthogonal lipid molecules (the alkyne-tagged fatty acid 17-ODYA, deuterium-labeled saturated fatty acid PA-D31, and unsaturated fatty acid OA-D34) to investigate the metabolic dynamics of lipid droplets (LDs) in live Caenorhabditis elegans (C. elegans). Using a hyperspectral SRS (hsSRS) microscope and subtraction method, the interfering non-Raman background was eliminated to improve the accuracy of lipid quantification. A linear relationship between SRS signals and fatty acid molar concentrations was accurately established. With this quantitative analysis tool, we imaged and determined the changes in concentration of the three fatty acids in LDs of fed or starved adult C. elegans. Using the hsSRS imaging mode, we also observed the desaturation of fatty acids in adult C. elegans via spectral analysis on the SRS signals from LDs. The results demonstrated the unique capability of hsSRS microscopy in quantitative analysis of lipid metabolism in vivo.